Streptococcus mutans is a microorganism that plays an important role in plaque formation in the oral cavity and is known to be most closely associated with tooth decay. The microorganism has the ability to degrade sugar to synthesize insoluble glucan in the extracellular matrix. The synthesized glucan is a sugar polymer having both a α-1,6 bond and a α-1,3 bond, forms a biofilm on the tooth surface due to its high stickiness and water insolubility, facilitates the adhesion of Streptococcus mutans, and aggregates various bacteria. Organic acids that are produced in this biofilm by adhesion of Streptococcus mutans and the like cause demineralization of tooth enamel or tissue to cause tooth decay. In addition, it is known that the formed biofilm also plays a major role in the development and progression of periodontitis.
Dental plaque comprises glucans that are polymers of glucose, and fructans that are polymers of fructose. Glucans are divided into three types: dextran having an alpha-1,6-glucose bond alone; a water-soluble glucan having an alpha-1,6-glucose bond as a main bond; and water-insoluble glucan (i.e., mutan) having an alpha-1,3-glucose bond as a main bond (Rolla et al., 1985). Mutan that is produced by Streptococcus mutans bacteria comprises 80-90% alpha-1,3-glucose bond and the remainder being an alpha-1,6-glucose bond.
Dextranase degrades the alpha-1,6-glucose bond of dental plaque. Known dextranase genes of fungal origin include genes from Penicillium, Paecilomyces, Aspergillus, Fusarium, Spicaria, Verticillium, Helminthosporium, Chaetomium, and the like, and known dextranase genes of bacterial origin include genes from Lactobacillus, Streptococcus, Cellvibrio, Cytophaga, Brevibacterium, Pseudomonas, Corynebacterium, Arthrobacter, Flavobacterium, and the like.
In foreign countries, Amano Co., Ltd. which is a Japanese enzyme company has launched a dextranase product derived from Paecilomyces lilacinus. The dextranase product is used mainly to remove dextran which is produced by contaminant bacteria in a process of producing sugar from sugarcane, and it is also used in some oral products.
Clinica Lion (product name) commercially available from Lion Co., Ltd. (Japan) also comprises dextranase containing triclosan. Although triclosan can temporally reduce Streptococcus mutans, it is expected that the dextranase enzyme alone will have an insufficient ability to degrade dental plaque.
In Korea, novel dextranase from Kimchi lactic acid bacteria was developed, and a toothpaste comprising dextranase and glucose oxidase was launched. However, they were not commercially successful, because the effect of dextranase on biofilm degradation is insufficient.
Alpha-1,3-glucanase is an enzyme that degrades the alpha-1,3-glucose bond of dental plaque and is also called mutanase. Enzymes that degrade alpha-1,3-glucose bonds have been found in Trichoderma sp. (Hasegawa et al., J. Biol. Chem., 244: 5460-5470, 1969), Streptomyces (Takehara et al., J Bacteriol., 145:729-735, 1981), Cladosporium resinae (Hare et al. Carbohydr Res., 58:415-432, 1977), Pseudomonas sp. (U.S. Pat. No. 4,438,093), Flavobacterium sp. (Japanese Patent No. 77038113), Bacillus circulanse (Japanese Patent No. 63301788), Aspergillus sp., and the like. However, studies on the ability to degrade mutan in the oral cavity remain at an early stage. Alpha-1,3-glucanase was developed overseas but was not commercialized due to its low mutan-degrading activity. Recently, alpha-1,3-glucanase from Bacillus circulans was developed in Japan and has been supplied to GlaxoSmithKline (GSK), Inc. and applied to Biotene mouthwashes and toothpaste products. However, the enzyme has not been launched as a product.
Accordingly, the present inventors have made extensive efforts to develop an enzyme having an excellent effect of degrading bacterial biofilms, and as a result, have found that novel alpha-1,3-glucanase from Trichoderma harzianum strain GF101 exhibits a high effect on biofilm degradation, thereby completing the present invention.